Megakaryocyte growth promoting activity protein

ABSTRACT

A novel human megakaryocytopoietic growth promoting activity factor capable of stimulating the growth of megakaryocytes and augments the differentiation or maturation of megakaryocytes. Also provided are processes for obtaining the factor in homogeneous form and producing it by recombinant genetic engineering techniques.

This is a continuation-in-part of pending U.S. patent application Ser.No. 07/332,651, filed on Apr. 3, 1989, now abandoned.

The present invention relates to a novel protein factor which stimulatesthe growth of megakaryocytes and augments the differentiation ormaturation of megakaryocytes. Also provided are processes for obtainingthe factor in homogeneous form and producing it by recombinant geneticengineering techniques.

BACKGROUND OF THE INVENTION

Megakaryocytes are the hematopoietic cells, largely found in the bonemarrow, but also in peripheral blood and perhaps other tissues as well,which produce platelets (also known as thrombocytes) and subsequentlyrelease them into circulation. Megakaryocytes, like all of thehematopoietic cells of the human hematopoietic system, ultimately derivefrom a primitive pluripotent marrow stem cell after passing through acomplex pathway comprising many cellular divisions and considerabledifferentiation and maturation. Mature megakaryocytes ultimately undergosubdivisions and release the cytoplasmic fragments which are circulatingplatelets.

The platelets derived from these megakaryocytic cells are critical forinitiating blood clot formation at the site of injury Platelets alsorelease growth factors at the site of clot formation that speed theprocess of wound healing and may serve other functions. Clinicalexperience has shown that control mechanisms exist to maintain effectiveplatelet numbers in humans, but that at times these specific controlsare either inadequate or ineffective and lead to depressed levels ofplatelets (thrombocytopenia) or thrombocytosis despite normal numbers ofred blood cells and white blood cells.

The inability to form clots is the most immediate and seriousconsequence of a low platelet count, a potentially fatal complication ofmany therapies for cancer. Such cancer patients are generally treatedfor this problem with platelet transfusions. Other patients frequentlyrequiring platelet transfusions are those undergoing bone marrowtransplantation or patients with aplastic anemia.

Platelets for such procedures are obtained by plateletphoresis fromnormal donors Like most human blood products, platelets for transfusionhave a relatively short shelf-life and also expose the patients toconsiderable risk of exposure to dangerous viruses, such as the humanimmunodeficiency virus (HIV) or the various hepatitis viruses.

The ability to stimulate endogenous platelet formation inthrombocytopenic patients with a concomitant reduction in theirdependence on platelet transfusion would be of great benefit. Inaddition the ability to correct or prevent thrombocytopenia in patientsundergoing radiation therapy or chemotherapy for cancer would make suchtreatments safer and possibly permit increases in the intensity of thetherapy thereby yielding greater anti-cancer effects.

For these reasons considerable research has been devoted to theidentification and purification of factors involved in the regulation ofmegakaryocyte and platelet production. Although there is considerablecontroversy, the factors regulating the growth and differentiation ofhematopoietic cells into mature megakaryocyte cells and the subsequentproduction of platelets by these cells are believed to fall into twoclasses.

Megakaryocyte colony-stimulating factors (meg-CSFs) are the first groupof regulatory factors which function to support the proliferation anddifferentiation of megakaryocytic progenitors (CFU-M) in culture. Thesecond group of factors have been defined by their activity towardsmegakaryocytes in either in vivo or in vitro bioassays. Factors whichelicit an in vivo response, such as an increase in the circulating levelof platelets have been defined as thrombopoietin ("TPO"). Factors whichsupport either the differentiation, maturation or development ofmegakaryocytes in an in vitro culture system have been termedmegakaryocyte stimulating activity, megakaryocyte potentiating activity,or thrombopoietin-like activity. It is unclear whether thrombopoieticfactors are structurally identical or related to any of the in vitrodefined megakaryocyte stimulating activities.

From the studies reported to date, it is not clear whether activitiesidentified as meg-CSF also have TPO activity or vice versa. Manydifferent reports in the literature describe factors which interact withcells of the megakaryocytic lineage and report megakaryocyte growthpromoting activities specific for the megakaryocyte lineage. [See, e.g.,E. Mazur, Exp. Hematol., 15:340-350 (1987); N. Williams et al, J. Cell.Physiol., 110:101-104 (1982); J. E. Straneva et al, Exp. Hematol.,14:919-929 (1986)]. An understanding of the specifics of positive andnegative control of megakaryocytopoiesis is incomplete.

For example, human IL-3 supports human megakaryocyte colony formationand, at least in monkeys, also frequently elicits an elevation inplatelet count. However, IL-3 influences hematopoietic cell developmentin all of the hematopoietic lineages and can be distinguished fromspecific regulators of megakaryocytopoiesis and platelet formation whichinteract selectively with cells of the megakaryocytic lineage.

There is strong evidence that in mice, murine IL-6 has thrombopoietinactivity in vivo and augments murine megakaryocyte colony formation withIL-3 in in vitro bioassays. However the thrombopoietic effect is notstriking (50-60% increase in circulating platelet numbers in 5 days) [T.Ishibashi et al, J. Clin. Invest., 79:286-289 (1987); T. Ishibashi etal, Blood, 74(4):1241-1244 (1989); T. Ishibashi et al, Proc. Natl. Acad.Sci. USA, 86:5953-5957 (1989)]. In vivo administration of IL-6 to micealso increases megakaryocyte size and ploidy. There is much lessevidence that IL-6 has TPO-like or megakaryocyte potentiating activityin human in vitro assays [see, e.g., E. Bruno and R. Hoffman, Exp.Hematol., 17:1038-4 (1989)]. In most of these assays human IL-6 hasshown no TPO-like activity [M. Teramura et al, Exp. Hematol.,17:1011-1016 (1989) and M. W. Long et al, J. Clin. Invest., 82:1779-1786(1988)].

R. Hoffman et al, J. Clin. Invest., 75:1174-1182 (1985) describes usinga human megakaryocyte colony assay to purify from serum a colonystimulating activity with an apparent MW of 46,000. This factor is foundin the 70-80% ammonium sulfate cut, binds to wheat germ lectin, andloses activity after deglycosylation. A similar activity was detected inthrombocytopenic rabbit plasma that increases the incorporation of ⁷⁵ Semethionine into platelets in mice. This activity was purified 7,000 foldfrom plasma, but contaminating proteins were present as determined bySDS-PAGE electrophoresis. See, e.g., R. Hill and J. Levin, Exp.Hematol., 14:752-759 (1986). Other serum derived factors are describedby J. E. Straneva et al, Exp. Hematol., 15:657-663 (1987); and E. Mazuret al, Exp. Hematol., 13:1164-1172 (1985].

Megakaryocyte growth promoting activities, and thrombopoietin also havebeen derived from human embryonic kidney (HEK) cells [See, e.g., T. P.McDonald, Exp. Hematol., 16:201-205 (1988); T. P. McDonald et al,Biochem. Med. Metab. Biol., 37:335-343 (1987); G. Tayrien and R. D.Rosenberg, J. Biol. Chem., 262:3262-3268 (1987) and others]. Each haspurified to homogeneity a 15,000 molecular weight activity that readilydimerizes to 30,000 molecular weight HEK-derived activity can increaseisotopic incorporation into platelets when given parenterally in mice,and increase the production of platelet factor 4-like proteins in rodentmegakaryocyte lineage cells. This activity is heat stable, and maintainsactivity after treatment with endoglycosidases, and binds to wheat germlectin.

Finally, activities have been described from urine that promotemegakaryocyte growth in rodents in vivo and in marrow culture. Kawakitahas partially purified an activity from urine that varies with patientplatelet count, and under dissociating conditions has a molecular weightof 45,000. The activity of this on human megakaryocyte progenitors hasnot been tested, nor has it been shown to be specific for themegakaryocyte hematopoietic lineage. [M. Kawakita et al, Br. J. Haem.,62:715-722 (1986); M. Kawakita et al, Blood, 61:556-560 (1983); see,also, S. Kuriya et al, Exp. Cell Biol., 55:257-264 (1987); K. Enomoto etal, Brit. J. Haem., 45:551-556 (1980)].

Despite such reports tentatively identifying such regulatory factors,the biochemical and biological identification and characterization ofthese factors has been hampered by the small quantities of the naturallyoccurring factors available from natural sources, e.g., blood and urine.

There remains a need in the art for the isolation, identification andproduction of additional proteins purified from their natural sources orotherwise produced in homogeneous form, which are capable of stimulatingor enhancing the production of platelets in vivo, to replace presentlyemployed platelet transfusions and otherwise useful in the treatmentand/or diagnosis of blood and blood platelet disorders.

BRIEF SUMMARY OF THE INVENTION

In one aspect the present invention provides a novel proteinaceousmegakaryocyte growth promoting activity factor ("MGPA") which issubstantially free from other human proteins. This protein may bepurified from cell sources producing the factor naturally or uponinduction with other factors. It may also be produced by recombinantgenetic engineering techniques. MGPA may also be synthesized by chemicaltechniques, or a combination of the above-listed techniques.

The MGPA of the present invention has been found to be specific to themegakaryocyte lineage, augmenting maturation and/or proliferation ofmegakaryocytes in the assay of Example 1 below.

Active MGPA has an apparent molecular weight of approximately 45 kd asdetermined by gel filtration chromatography and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. MGPA is further characterized inthat it does not bind wheat germ lectin. The factor does bind to acation exchange resin (e.g., Pharmacia Mono S column) at acidic pH, butdoes not bind to an anion exchange resin at neutral pH. MGPA is foundconsistently in the 30-50% ammonium sulphate precipitate ofthrombocytopenic patient plasma, but cannot be detected in normal humanplasma. MGPA is present in normal urinary protein concentrates.

MGPA is further characterized by its ability to act in an additive orsynergistic manner with GMCSF and IL3 to promote megakaryocyte growth inliquid bone marrow culture systems.

Still a further aspect of the present invention is a process forisolating and purifying the MGPA composition of the present invention ora fragment thereof from human urine. This purification process providedby the present invention involves the following steps. The first twosteps are ammonium sulfate precipitation, followed by cation exchangecolumn chromatography on sulphopropyl Sephadex at pH5.4 in 25 mMammonium acetate buffer. This step is followed by subjecting theMGPA-containing fractions to filtration through a polyethyleneimineanion exchange membrane in 50 mM ammonium bicarbonate buffer at pH 7.4.The MGPA-containing filtrate is then subjected to reverse phase highperformance liquid chromatography (HPLC) on a C3 column with 0.05%trifluoroacetic acid (TFA) and acetonitrile as the mobile phase solvent.

A further aspect of the present invention is homogeneous MGPA purifiedfrom urine or produced via recombinant or synthetic techniques which ischaracterized by a specific activity in the radioimmunoassay of greaterthan 1×10⁶ units/mg.

Another aspect of the present invention is a DNA sequence that encodesthe expression of a human MGPA protein. This DNA sequence may include anisolated DNA sequence that encodes the expression of a human MGPAprotein as described above. The DNA sequence may also include 5' and 3'human non-coding sequences flanking the MGPA coding sequence. The DNAsequence may also encode an amino terminal signal peptide.

Also provided by the present invention is a recombinant DNA moleculecomprising vector DNA and a DNA sequence encoding human MGPA. The DNAmolecule provides the MGPA DNA in operative association with aregulatory sequence capable of directing the replication and expressionof MGPA in a selected host cell. Host cells transformed with such DNAmolecules for use in expressing recombinant MGPA protein are alsoprovided by the present invention.

The DNA molecules and transformed cells of the invention are employed inanother aspect, a novel process for producing recombinant human MGPAprotein, or peptide fragments thereof. In this process a cell linetransformed with a DNA sequence encoding expression of MGPA protein or afragment thereof (or a recombinant DNA molecule as described above) inoperative association with a suitable regulatory or expression controlsequence capable of controlling expression of the protein is culturedunder appropriate conditions permitting expression of the recombinantDNA. This claimed process may employ a number of known cells as hostcells for expression of the protein. Presently preferred cell lines forproducing MGPA are mammalian cell lines and bacterial cells.

The expressed MGPA protein is then harvested from the host cell, celllysate or culture medium by suitable conventional means. The conditionedmedium may be processed through the same purification steps ormodifications thereof as used to isolate the MGPA from urine.

As still a further aspect of the present invention, there is providedrecombinant MGPA protein. This protein is substantially free from otherhuman proteinaceous materials and comprising a DNA sequence encoding oneor more of the peptide fragments or sequences described herein. The MGPAprotein of this invention is also characterized by containing one ormore of the physical, biochemical, pharmacological or biologicalactivities described herein.

Another aspect of this invention provides pharmaceutical compositionscontaining a therapeutically effective amount of homogeneous orrecombinant MGPA or an effective amount of one or more active peptidefragments thereof. These pharmaceutical compositions may be employed inmethods for treating disease states or disorders characterized by adeficiency or defect of platelets.

Thus the MGPA composition of the present invention or pharmaceuticallyeffective fragments thereof may be employed in the treatment of aplasticanemias, e.g., to augment production of platelets in patients havingimpaired platelet production (such as AIDS patients or patientsundergoing cancer chemotherapy). The MGPA may be used to treat blooddisorders such as thrombocytopenia. MGPA may be used as an adjunctivetherapy for bone marrow transplant patients.

A further aspect of the invention, therefore, is a method for treatingthese and other pathological states resulting from a deficiency ofplatelets by administering to a patient a therapeutically effectiveamount of MGPA or one or more peptide fragments thereof in a suitablepharmaceutical carrier. These therapeutic methods may includeadministering simultaneously or sequentially with MGPA or one or morepeptide fragments thereof an effective amount of at least one othermeg-CSF or TPO-like factor, a cytokine, hematopoietin, interleukin,growth factor, or antibody.

Still another aspect of the present invention are antibodies directedagainst human MGPA or a fragment thereof. As part of this aspect,therefore, the invention claims cell lines capable of secreting suchantibodies and methods for their production and use in diagnostic ortherapeutic procedures.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description of preferredembodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

The novel human megakaryocyte growth promoting activity factor, MGPA,provided by the present invention is a homogeneous protein orproteinaceous composition substantially free of association with otherhuman proteinaceous materials. This protein can be produced viarecombinant techniques to enable large quantity production of pure,active MGPA useful for therapeutic applications. Alternatively thisprotein may be obtained as a homogeneous protein purified from humanurine or from a mammalian cell line secreting or expressing it. FurtherMGPA or active fragments thereof may be chemically synthesized.

MGPA of the present invention is characterized by one or more of thefollowing biochemical and biological properties:

(1) The composition of the present invention has an apparent molecularweight of approximately 45 kd as determined by 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under either non-reducingor reducing conditions.

(2) The composition of the present invention has an apparent molecularweight of approximately 40-50 kd on gel filtration chromatography.

(3) The composition of the present invention has a specific activity inthe megakaryocyte growth promoting assay of Example 1 of greater thanapproximately 1×10⁶ units/mg protein.

(4) The MGPA composition of the present invention is capable of bindinga cation exchange column under acidic conditions of pH 5.4.

(5) The MGPA composition of the present invention is not capable ofbinding to Wheat Germ lectin.

(6) The MGPA composition of the present invention does not bind to ananion exchange resin at neutral pH.

(7) The MGPA composition of the present invention is found consistentlyin the 30-50% ammonium sulfate precipitate of thrombocytic patienturine.

(8) The MGPA composition of the present invention cannot be detected innormal human plasma with the current assays.

The biological activity of the MGPA composition of the present inventionis demonstrated by its ability to stimulate the growth and developmentof megakaryocytes in the radioimmunological megakaryocyte growthpromoting assay of Example 1. This in vitro assay for regulatoryactivities stimulated by low platelet counts detects cell-boundGPIIb/IIIa, a megakaryocyte lineage specific glycoprotein which isexpressed on small morphologically unrecognizable megakaryocyteprecursors as well as recognizable megakaryocytes and platelets. Theassay enables quantitative assessment of in vitro megakaryocytopoiesis[B. W. Grant et al, Blood, 69:1334-1339 (1987)]. This assay is describedin detail in Example 1 below.

MGPA was originally detected in the citrated plasma of human patientswith aplastic anemia. Unfractionated plasma from these patientsdemonstrate an enhanced support of megakaryocyte growth in vitro.Plasmas from other thrombocytopenic patients have been shown to containMGPA. Human MGPA was initially purified from this human plasma by asequence of purification steps and techniques specifically described inExample 2 below. However, this factor may also be purified fromthrombocytopenic patient urine.

Two cellular sources of MGPA have presently been identified.Osteosarcoma cells explanted from a tumor and passaged in culture,called HS10, elaborate MGPA. MGPA has not been detected in conditionedmedia when HS10 cells are grown in the presence of serum. When withoutserum, these cells produce an activity very similar to the plasma MGPAwith an unfractionated conditioned medium having 100 to 200 units MGPAper A₂₈₀ unit. This activity co-purifies with the plasma MGPA on anACA34 gel filtration column. Because HS10 is not a transformed cellline, it grows slowly, dies off, and secretes very little MGPA (serumfree conditioned media has about 1/10 of a unit per milliliter). Thesecells may be transformed to produce MGPA consistently. Alternatively,other osteosarcoma cell lines may be detected with similar MGPAproduction.

MGPA similar to that found in plasma has also been detected inconditioned media from human umbilical vein endothelial (HUV) cells.Conditioned media from these cells (supplied by Dr. Faller, Dana-Farberand Dr. Shorer, University of Minnesota) have been found to promotemegakaryocyte growth in the radioimmunoassay of Example 1. This materialco-purifies with plasma MGPA by gel filtration, and supportsmegakaryocyte growth in phytohemagglutinin-leukocyte conditioned medium(PHALCM), IL3 or GM-CSF in a dose dependent way similar to the plasmaand urinary MGPA. This activity is not inhibited by anti GM-CSF.

The purification techniques employed in obtaining MGPA from human urinecomprises the following steps which are outlined in detail in Example 3.These steps include subjecting the unconcentrated urine to ammoniumsulfate precipitation; binding the 80% ammonium sulfate fraction to acation exchange chromatographic column (sulfopropyl Sephadex) in 25 mMammonium acetate, pH 5.4 and eluting the bound protein in a gradient ofNaCl; passing the 0.15M NaCl eluate containing MGPA through an anionexchange membrane (polyethyleneimine) in ammonium bicarbonate buffer, pH7.4; and finally applying the filtrate through a cycle of reverse phaseHPLC using H₂ O/TFA/acetonitrile as the solvent.

Fractionation techniques that have not been useful includehydroxyapatite, heparin sepharose and wheat germ agglutinin using twodifferent lectin bead preparations [Sigma].

Homogeneous MGPA may be obtained by applying the above purificationprocedures, which are described in detail in Example 3, to humanthrombocytopenic urine or other sources of human MGPA e.g., cell ortissue sources. Procedures for culturing a cell (or tissue) source whichmay be found to produce MGPA are known to those of skill in the art.

MGPA of this invention differs from other TPO-like factors of the priorart. For example, MGPA differs from TSF [MacDonald, supra] and megCSA[Hoffman, supra] in apparent molecular weight, in the % ammonium sulfatethat precipitates it from plasma, in its behavior on ion exchangecolumns, and in its lack of binding to wheat germ lectin.

MGPA or one or more peptide fragments thereof may also be produced viarecombinant techniques. To obtain the DNA sequence for MGPA, thepurified MGPA material is reduced and digested with trypsin. Trypticfragments are isolated and sequenced by conventional techniques.Oligonucleotide probes are synthesized using the genetic code to predictall possible sequences that encode the amino acid sequences of thetryptic fragments. Several sequences are generated as probes. The MGPAcDNA is identified by using these probes to screen a human genomiclibrary. Alternatively, the mRNA from a cell source of MGPA can be usedto make a cDNA library which can be screened with the probes to identifythe cDNA encoding the MGPA polypeptide.

Using these probes to screen human genomic library, a cDNA clone isobtained. To obtain a full length clone, the obtained cDNA sequences maybe employed as probes to rescreen the library and hybridize to the fulllength MGPA sequence.

The human CDNA for MGPA can also be obtained by subcloning a full lengthhuman genomic clone into an expression vector, transfecting it into COScells, preparing a cDNA library from these transfected COS cells andscreening by hybridization for MGPA cDNA. Once the entire cDNA isidentified, it or any portion of it that encodes an active fragment ofMGPA, can be introduced into any one of a variety of expression vectorsto make an expression system for MGPA or one or more fragments thereof.

By such use of recombinant techniques, DNA sequences encoding the MGPApolypeptide are obtained. The present invention also encompasses theseDNA sequences, free of association with DNA sequences encoding otherproteins, and coding on expression for MGPA polypeptides. These DNAsequences include those sequences encoding all or a fragment of MGPA andthose sequences which hybridize under stringent hybridization conditions[see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual). ColdSpring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences.

An example of one such stringent hybridization condition ishybridization in 4XSSC at 65° C., followed by a washing in 0.1XSSC at65° C. for an hour. Alternatively an exemplary stringent hybridizationcondition is in 50% formamide, 4XSSC at 42° C.

DNA sequences which hybridize to the sequences for MGPA under relaxedhybridization conditions and which code on expression for MGPA peptideshaving MGPA biological properties also encode novel MGPA polypeptides.Examples of such non-stringent hybridization conditions are 4XSSC at 50°C. or hybridization with 30-40% formamide at 42° C. For example, a DNAsequence which shares regions of significant homology, e.g., sites ofglycosylation or disulfide linkages, with the sequences of MGPA andencodes a protein having one or more MGPA biological properties clearlyencodes a MGPA polypeptide even if such a DNA sequence would notstringently hybridize to the MGPA sequences.

Allelic variations (naturally-occurring base changes in the speciespopulation which may or may not result in an amino acid change) of DNAsequences encoding the peptide sequences of MGPA are also included inthe present invention, as well as analogs or derivatives thereof.Similarly, DNA sequences which code for MGPA polypeptides but whichdiffer in codon sequence due to the degeneracies of the genetic code orvariations in the DNA sequence of MGPA which are caused by pointmutations or by induced modifications to enhance the activity, half-lifeor production of the polypeptides encoded thereby are also encompassedin the invention.

MGPA polypeptides may also be produced by known conventional chemicalsynthesis. Methods for constructing the polypeptides of the presentinvention by synthetic means are known to those of skill in the art. Thesynthetically-constructed MGPA polypeptide sequences, by virtue ofsharing primary, secondary, or tertiary structural and conformationalcharacteristics with MGPA polypeptides may possess MGPA biologicalproperties in common therewith. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedMGPA polypeptides in therapeutic and immunological processes.

Modifications in the peptides or DNA sequences encoding MGPA can be madeby one skilled in the art using known techniques. Modifications ofinterest in the MGPA sequences may include the replacement, insertion ordeletion of a selected amino acid residue in the coding sequences.Mutagenic techniques for such replacement, insertion or deletion arewell known to one skilled in the art. [See, e.g., U.S. Pat. No.4,518,584.]

Specific mutations of the sequences of the MGPA polypeptide may involvemodifications of a glycosylation site, if any. The absence ofglycosylation or only partial glycosylation results from amino acidsubstitution or deletion at any asparagine-linked glycosylationrecognition site or at any site of the molecule that is modified byaddition of O-linked carbohydrate. An asparagine-linked glycosylationrecognition site comprises a tripeptide sequence which is specificallyrecognized by appropriate cellular glycosylation enzymes. Thesetripeptide sequences are either Asp-X-Thr or Asp-X-Ser, where X can beany amino acid. A variety of amino acid substitutions or deletions atone or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Expression of such altered nucleotide sequences producesvariants which are not glycosylated at that site.

Other analogs and derivatives of the sequence of MGPA which would beexpected to retain MGPA activity in whole or in part may also be easilymade by one of skill in the art given the disclosures herein. One suchmodification may be the attachment of polyethylene glycol (PEG) ontoexisting lysine residues in the MGPA sequence or the insertion of one ormore lysine residues or other amino acid residues that can react withPEG or PEG derivatives into the sequence by conventional techniques toenable the attachment of PEG moieties. Such modifications are believedto be encompassed by this invention.

The present invention also provides a method for producing MGPApolypeptides or active fragments thereof. One method of the presentinvention involves introducing the cDNA encoding a MGPA polypeptide intoan expression vector to make an expression system for MGPA. A selectedhost cell is transformed with the vector and cultured. The method ofthis present invention therefore comprises culturing a suitable cell orcell line, which has been transformed with a DNA sequence coding onexpression for a MGPA polypeptide under the control of known regulatorysequences. Regulatory sequences include promoter fragments, terminatorfragments and other suitable sequences which direct the expression ofthe protein in an appropriate host cell. The expressed factor is thenrecovered, isolated and purified from the culture medium (or from thecell, if expressed intracellularly) by appropriate means known to one ofskill in the art.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO) or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625(1981), or alternatively, Kaufman et al, Mol. Cell. Biol.,5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446. Othersuitable mammalian cell lines, are the monkey COS-1 cell line, and theCV-1 cell line. Further exemplary mammalian host cells includeparticularly primate cell lines and rodent cell lines, includingtransformed cell lines. Normal diploid cells, cell strains derived fromin vitro culture of primary tissue, as well as primary explants, arealso suitable. Candidate cells may be genotypically deficient in theselection gene, or may contain a dominantly acting selection gene. Othersuitable mammalian cell lines include but are not limited to, HeLa,mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHKor HaK hamster cell lines.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, MC1061 and strains used in the following examples) are well-knownas host cells in the field of biotechnology. Various strains of B.subtilis, Pseudomonas, other bacilli and the like may also be employedin this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

The present invention also provides recombinant molecules or vectors foruse in the method of expression of novel MGPA polypeptides. Thesevectors contain the MGPA DNA sequences and which alone or in combinationwith other sequences code for MGPA polypeptides of the invention oractive fragments thereof. Alternatively, vectors incorporating modifiedsequences as described above are also embodiments of the presentinvention and useful in the production of MGPA polypeptides. The vectoremployed in the method also contains selected regulatory sequences inoperative association with the DNA coding sequences of the invention andcapable of directing the replication and expression thereof in selectedhost cells.

One vector which is described in the examples below is pXM, which isparticularly desirable for expression in COS cells [Y. C. Yang et al,Cell, 47:3-10 (1986)]. Another vector which is desirable for expressionin mammalian cells, e.g., CHO cells, and is described in the examples ispEMC2B1. Mammalian cell expression vectors described herein may besynthesized by techniques well known to those skilled in this art. Thecomponents of the vectors, e.g. replicons, selection genes, enhancers,promoters, and the like, may be obtained from natural sources orsynthesized by known procedures. See, Kaufman et al, J. Mol. Biol.,159:511-521 (1982); and Kaufman, Proc. Natl. Acad. Sci., USA, 82:689-693(1985). Alternatively, the vector DNA may include all or part of thebovine papilloma virus genome [Lusky et al, Cell, 36:391-401 (1984)] andbe carried in cell lines such as C127 mouse cells as a stable episomalelement. The transformation of these vectors into appropriate host cellscan result in expression of the MGPA polypeptides.

Other appropriate expression vectors of which numerous types are knownin the art for mammalian, insect, yeast, fungal and bacterial expressioncan also be used for this purpose.

Thus MGPA or active fragments thereof, purified to homogeneity from cellsources or produced recombinantly or synthetically, may be used in apharmaceutical preparation or formulation to stimulate platelet recoveryin patients suffering from thrombocytopenias associated with marrowhypoplasia, e.g., aplastic anemia following chemotherapy or bone marrowtransplantation. Other platelet disorders for which treatment with MGPAmay be useful include diseases of increased platelet consumption andproduction, such as, disseminated intravascular coagulation, immunethrombocytopenia, and thrombotic thrombocytopenia. Additionally MGPA maybe useful in treating myeloproliferative thrombocytotic diseases, andthrombocytosis in inflammatory conditions and in iron deficiency.

Still another use for MGPA or fragments thereof is in the treatment ofdisorders resulting from defects in platelets or damage to platelets,e.g. resulting from transient poisoning of platelets by other chemicalor pharmaceutical agents or therapeutic manipulations. MGPA may beemployed to stimulate the "shedding" of new "undamaged" platelets insuch patients.

Therapeutic treatment of such platelet disorders or deficiencies withthese MGPA polypeptide compositions may avoid undesirable side effectscaused by treatment with presently available serum-derived factors ortransfusions of human platelets. It may also be possible to employ oneor more peptide fragments of MGPA in such pharmaceutical formulations.

The polypeptides of the present invention may also be employed, alone orin combination with other cytokines, hematopoietins, interleukins,growth factors or antibodies in the treatment of the above-identifiedconditions.

Therefore, as yet another aspect of the invention are therapeuticcompositions for treating the conditions referred to above. Suchcompositions comprise a therapeutically effective amount of the MGPApolypeptide or a therapeutically effective fragment thereof in admixturewith a pharmaceutically acceptable carrier. This composition can besystematically administered parenterally. Alternatively, the compositionmay be administered intravenously. If desirable, the composition may beadministered subcutaneously. When systematically administered, thetherapeutic composition for use in this invention is in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such pharmaceutically acceptable protein solutions, having due regardto pH, isotonicity, stability and the like, is within the skill of theart.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician consideringvarious factors which modify the action of drugs, e.g. the condition,body weight, sex and diet of the patient, the severity of any infection,time of administration and other clinical factors. Generally, the dailyregimen should be in the range of 1-1000 micrograms of MGPA protein orfragment thereof or 50 to 5000 units of protein per kilogram of bodyweight.

The therapeutic method, compositions and polypeptides of the presentinvention may also be employed, alone or in combination with othercytokines, hematopoietins, interleukins, growth factors or antibodies inthe treatment of disease states characterized by other symptoms as wellas platelet deficiencies. It is anticipated that this molecule, willprove useful in treating some forms of thrombocytopenia in combinationwith general stimulators of hematopoiesis, such as lL-3 or GM-CSF. Othermegakaryocytic stimulatory factors, e.g., meg-CSF, or other moleculeswith TPO-like activity may also be employed with MGPA. Additionalexemplary cytokines or hematopoietins for such co-administration includeG-CSF, CSF-1, IL-1, IL-4, M-CSF, IL-7, or erythropoietin. The dosagerecited above would be adjusted to compensate for such additionalcomponents in the therapeutic composition. Progress of the treatedpatient can be monitored by conventional methods.

Other uses for these novel polypeptides are in the development ofantibodies generated by standard methods for in vivo or in vitrodiagnostic or therapeutic use. Such antibodies may include bothmonoclonal and polyclonal antibodies, as well as chimeric antibodies or"recombinant" antibodies generated by known techniques. Also provided bythis invention are the cell lines generated by presenting MGPA or afragment thereof as an antigen to a selected mammal, followed by fusingcells of the animal with certain cancer cells to create immortalizedcell lines by known techniques. The methods employed to generate suchcell lines and antibodies directed against all or portions of a humanMGPA polypeptide of the present invention are also encompassed by thisinvention.

The antibodies of the present invention may be utilized for in vivo andin vitro diagnostic purposes, such as by associating the antibodies withdetectable labels or label systems. Alternatively these antibodies maybe employed for in vivo and in vitro therapeutic purposes, such as byassociation with certain toxic or therapeutic compounds or moietiesknown to those of skill in this art.

The following examples illustratively describe the purification andcharacteristics of homogeneous human MGPA and other methods and productsof the present invention. These examples are for illustration and do notlimit the scope of the present invention.

EXAMPLE 1 Radioimmunological Megakaryocyte Growth Promoting Assay

The following assay to detect cell-bound GPIIb/IIIA allows quantitativeassessment of in vitro megakaryocytopoiesis. This system uses aradioimmunoassay to measure the generation of megakaryocytes in culturesof normal human bone marrow and is reproducible and reliable as a screenfor megakaryocyte growth.

Non-adherent mononuclear bone marrow cells are cultured for two weeks inIscove's modified Dulbecco's medium, fetal calf serum or other serum orplasma, and other growth factors. Aliquots of cultured cells are washedfree of culture media, and exposed to ¹²⁵ I-HPIID, a murine monoclonalantibody specific to the GPIIb/IIIA complex [see, Grant et al, in"Megakaryocyte Development and Function", eds, Allen R. Liss, pp.117-121 (1986)]. The binding of iodinated antibody is specific tomegakaryocytes, and when free antibody is washed away from the cellpellet, radioactivity bound in the pellet is a quantitative measure ofthe number of megakaryocytes present.

The quantitative nature of the assay has been demonstrated bycorrelation with the numbers of morphologically identifiablemegakaryocytes in cytospin preparations from individual wells of theMGPA assay. This assay detects small, morphologicallydifficult-to-recognize megakaryocytes as well as larger, more maturemegakaryocytes. Thus it is useful in screening for megakaryocyte growthfactors affecting viability, proliferation or maturation at any point indifferentiation. This assay cannot distinguish between increases innumbers of cells, increases in size of cells, or increases in thedensity of GPIIb/IIIA on the cell surface. Therefore, the megakaryocytemorphology and the number and variety of other cells in wells ofinterest are further evaluated using cytocentrifuge preparations.

This assay correlates well with human megakaryocyte colony assays forresponse to class I myeloid growth factors (IL-3 and GM CSF), and humanaplastic plasma. Optimal growth and maximum signal occurs with 3-4×10⁵cells per well and an incubation period of 12-14 days. A useful test toconfirm or rule out the specificity of individual megakaryocyte growthpromoting activities is quantitation of the percent megakaryocytes amongcultured cells by immunoalkaline phosphatase stain of GPIIb/IIaexpressing cells.

This assay system has readily adapted to the screening of fractionatedmaterial for MGPA. For example, after isolation procedures, as describedin Example 3, fractions are tested for protein content by absorbtion atA₂₈₀, dialyzed against ammonium bicarbonate buffer (50mM), lyophilized,resuspended in Iscoves medium with 5% fetal calf serum, and filteredthrough 0.2 micron filters. Each fraction is tested at 3-6 dose levels(dilutions) with two different normal bone marrows with the MGPA assayfollowed by cytospin analysis and other tests on the marrows, asindicated.

For example, routine screening of a fraction for MGPA is performed usinghuman marrow cultured in 30% fetal calf serum in the presence ofsynergizing Class I growth factors, usually 5%Phytohemagglutinin-Leukocyte Conditioned Medium (PHALCM) or 10 units ofrecombinant IL-3 [Genetics Institute, Inc., Cambridge, Mass. ] added tosupport the early stem cells and maximize expression of megakaryocytespecific stimulating activities. Optimal growth for each marrow isassessed in wells supplemented with aplastic plasma and PHALCM, and allwells are assayed in duplicate or triplicate in the radioimmunoassay.

As used throughout this specification, one unit of MGPA is defined asthe amount of MGPA required to stimulate megakaryocyte growth to twicethat of the background (the fetal calf serum plus PHALCM control) inthis assay. Thus, the number of units in a sample equals the differencebetween the CPM bound at the end of culture in the sample well minus theCPM bound in the fetal calf serum plus PHALCM control, divided by theCPM bound in that same control. The calculation of the number of unitsin a given fraction is done by averaging the calculated unitage fromwells representing the linear part of the dose response curve from twoindependent experiments using two different marrow samples.

The screening assay system is optimized to detect factors that completethe megakaryocyte developmental program promoted by Class Ihemopoietins.

EXAMPLE 2 Isolation of Megakaryocyte Growth Promoting Activity fromPlasma

The original isolation of MGPA from citrated plasma from patients withaplastic anemia employed the following purification steps:

(1) Ammonium sulfate precipitation (30-50% pellet, 5 fold purification);

(2) ACA54 or ACA34 gel filtration (10 fold purification);

(3) sulfopropyl sephadex (SPC-50) ion exchange chromatography (in 25 mMammonium acetate, eluted with sodium chloride, 10 fold purification);and

(4) preparative SDS gel electrophoresis (8% polyacrylamide matrix inLaemmli running buffer, 0.01% SDS).

Plasma MGPA was purified through step 3 above 600 times relative towhole aplastic plasma. The apparent MW as determined by the gelfiltration and SDS PAGE steps was found to be approximately 40-50 kd.

To establish that the MGPA initially partially purified from aplasticanemia plasma was an activity common to other patients withthrombocytopenia, plasma from four patients with aplastic anemia, fourpatients with immune thrombocytopenia, three patients with chemotherapyinduced thrombocytopenia, two patients with thrombotic thrombocytopenicpurpura and one patient with megakaryocytic thrombocytopenia were testedfor MGPA.

Each thrombocytopenic plasma yielded maximum bioactivity in theradioimmunological megakaryocyte growth promoting assay of Example 1 inthe 30-50% ammonium sulfate precipitate. The molecular weight range ofthe activity in those fractions further fractionated by gel filtrationwas consistent. Pooled fractions including relative molecular weightsbetween 30-50 kd from these eight plasmas each showed titratable MGPA.

No titratable MGPA was recovered from similar fractionations of threenormal human plasmas, of fetal calf serum, nor of plasma from threepatients with thrombocytosis.

EXAMPLE 3 Isolation of MGPA from Urine

MGPA is purified from fresh patient urine using the following sequentialfractionation steps: ammonium sulfate precipitation, sulfopropylsephadex (SPC-50) cation exchange chromatography, polyethyleneimine(PEI) anion exchange membrane filtration, and reverse phase highperformance liquid chromatography (HPLC). A summary of this purificationscheme is presented in Table I.

The first step employed in the purification is an 80% ammonium sulfateprecipitation of protein from unconcentrated urine. The purification ofMGPA is one-fold, yielding a fraction with a specific activity of 500units per milligram protein. When normal human urine is subjected toammonium sulfate precipitation, MGPA activity in the 80% proteinprecipitate has approximately 40 units per mg protein. Compared withnormal control urine, the level of MGPA present in thrombocytopenicurine is increased by greater than 5-12 fold.

The second purification step is the use of cation exchange columnchromatography. The MGPA-containing ammonium sulfate fraction isdialyzed into 25 mM ammonium acetate pH 5.4 and then applied to asulfopropyl sephadex (SPC-50) ion exchange column equilibrated in thesame buffer. Bound protein is eluted in a gradient of NaCl from 0-1M.MGPA elutes at approximately 150 mM NaCl resulting in approximately a60-fold enrichment of MGPA specific activity.

Further purification of the MGPA is achieved by the third step in whichthe 150 mM eluate from the SPC-50 column is subjected to anion exchangefiltration. MGPA-containing protein is dialyzed into 50 mM ammoniumbicarbonate pH 7.4 and passed through a polyethyleneimine (P-I) anionexchange membrane equilibrated in the same buffer. MGPA activity iscollected in the flow-through fraction. This step enriches MGPA specificactivity by approximately 10-fold.

The fourth step in the purification is achieved using reverse phase highperformance liquid chromatography (HPLC) on a C3 column. The PEImembrane filtrate which contains MGPA is dialyzed into 0.05% TFA andloaded onto a C3 column equilibrated in 0.05% TFA. Bound protein iseluted in a gradient of acetonitrile in 0.05% TFA. MGPA elutes between62 and 70% acetonitrile. At this point in the purification MGPA has aspecific activity of greater than 1×10⁶ units per mg protein. Based onSDS polyacrylamide gel electrophoresis (SDS-PAGE), the major proteinband at 45,000 under reducing conditions contains the MGPA activity whenit is excised from the gel and eluted into a suitable buffer for assay.The purified MGPA obtained from the HPLC step may be suitable forprotein sequencing directly or it may be subjected to SDS PAGE prior toprotein sequencing to remove minor contaminants which may be presentafter the fourth step.

                  TABLE I                                                         ______________________________________                                        Purification of MGPA from Thrombocytopenic Patient Urine                      Purification Step        Total Fold                                                                              Total                                      (%)          .sup.a Units/mg.sup.b                                                                     Purification                                                                            Yield                                      ______________________________________                                        Ammonium Sulfate                                                                           5 × 10.sup.2                                                                          1       100                                        precipitation                                                                 SPC-50       3 × 10.sup.4                                                                          60      13                                         PEI          4 × 10.sup.5                                                                         800      13                                         C3 65-70%    1.1 × 10.sup.7                                                                      22000      8                                         ______________________________________                                         .sup.a Units are defined as in Example 1.                                     .sup.b Mgs assigned by absorbance at 280 nm.                             

Urinary MGPA is markedly increased in thrombocytopenic patients. Inunfractionated urinary proteins where a mixture of enhancing andinhibitory factors are observed to be present, greater than 5-15 foldmore activity in patient urine is observed than in normal human urine.This is strong evidence that the activity measured in the assay ofExample 1 is a physiologic regulator of megakaryocyte production that isconstitutively produced and measurable in normal urine, but markedlyinduced and thus measurable in the plasma as well as the urine ofthrombocytopenic patients.

EXAMPLE 4 Biological Activities of Human MGPA

The following assays were performed using the purified MGPA from plasmaor urine as described. After the first few stages of purification, thebiological characteristics of plasma MGPA and urinary MGPA areidentical. The recombinant version of the molecule is expected toexhibit the same biological properties in these same assays or otherassays

(1) The Class I hemopoietins IL3 and GM-CSF maintain viability andsupport proliferation of undifferentiated myeloid precursor cells andpromote megakaryocyte growth in the assay system of Example 1. Todemonstrate that MGPA is not IL-3 or GM-CSF, plasma MGPA partiallypurified by ammonium sulfate precipitation and AcA 34 gel filtration wasadded to megakaryocyte growth supported by both IL-3 and GMCSF in a dosedependent manner. PHALCM was also employed as a crude source of theseactivities to provide maximal Class I activity to the cells to beassayed.

Neither IL3 nor GMCSF increase megakaryocyte growth in liquid cultureover that of other myeloid cells. Megakaryocyte growth supported by MGPAis not neutralized by antibodies to IL-3 [Genetics Institute] if theculture is supplemented with GM-CSF to support stem cell growth.Similarly, megakaryocyte growth supported by MGPA is not neutralized byantibodies to GM-CSF if IL-3 is supplied. A combination of theseantisera reverses all megakaryocyte growth stimulated by PHALCM. Humanbone marrow grown in the presence of IL3 and GMCSF produced similaramounts of megakaryocytes in the presence or absence of antiserumagainst GMCSF. When human marrow was grown in methylcellulose, very fewgranulocyte/ macrophage colonies are stimulated above FCS control with 3units/ml of partially purified MGPA.

MGPA adds to the effect of PHALCM in the marrow cultures. On day 14 ofculture very little dose dependent megakaryocyte growth is seen incultures supplemented with FCS and HPLC-purified MGPA as the onlyexogenous growth factors. Marked dose dependent enhancement ofmegakaryocyte growth by MGPA is observed in the presence of PHALCM,IL-3, and GM-CSF. Each of these sources of class I hemopoietins appearsto support the same or very similar groups of MGPA responsive cells, asthe response curves are quite similar from most marrows.

Based on these results it is expected that MGPA will produce an additiveor synergistic effect with either IL3 and/or GM-CSF in therapeuticapplications.

(2) That MGPA stimulates megakaryocyte growth has also been shown bytesting MGPA in the human megakaryocyte colony assays of Mazur et al,Blood, 57:277-286 (1981) and Solberg et al, in The Inhibitors ofHematopoiesis, pp. 111-121 (1987). MGPA alone fails to supportmegakaryocyte colony growth from either human marrow or peripheral bloodin these systems. MGPA markedly increases the number of megakaryocytecolonies supported by IL-3, consistent with the concept that MGPA actsto complete the differentiation supported by IL-3.

(3) Similarly in murine systems, MGPA promoted megakaryocyte maturationin the serum-free Acetylcholinesterase assay of Burstein, cited above.

(4) MGPA only supported murine megakaryocyte colony formation in thepresence of IL-3 or WEHI conditioned medium in a megakaryocyte colonyassay using murine bone marrow in a fibrin clot [S. Kuriya et al, Exp.Hematol., 15:896-901 (1987)].

(5) Cytospin preparations of MGPA supported cultures confirmed that MGPAnot only increases the growth of megakaryocytes but also increases thenumber of megakaryocytes relative to other cells, as would be found witha specific promoter of megakaryocyte growth. To test for this, partiallypurified MGPA from SPC-50 was titrated in FCS, alone, or in 5% PHALCM.The percent megakaryocytes was determined on cytospin preparations ofcells from the liquid marrow cultures using an immunoalkalinephosphatase stain with antibody against GPIIb/IIIa. 1000 cells werecounted on each slide. At each dose of MGPA tested, the percent ofmegakaryocytes correlated well with the total CPM bound in the assay ofExample 1. When the MGPA plus PHALCM treated culture, the IL-3 treatedculture and GM-CSF treated culture are compared within the optimal doserange for MGPA, the actual percentage of megakaryocytes decreases inboth the IL-3 and GM-CSF treated samples at higher concentrations due toan increase in number of other myeloid cells that grow in response tothese factors.

The megakaryocytes supported by MGPA in culture are consistently moremature and somewhat larger in size than those grown in control wells(FCS with PHA-LCM) suggestive of a maturational effect. Marrow supportedby FCS and PHA-LCM has produced recognizable megakaryocytes ranging instage from I to II/III to IV. A cytospin from the same marrow grownunder identical conditions except that HPLC-purified MGPA was added showthat not only are more megakaryocytes recognizable per field, but theytend to be more mature and have far denser cytoplasm. No increase isseen in the number of other myeloid cells.

(6) The following tests for stability were performed on both urinary andplasma purified MGPA and have revealed the following characteristics ofMGPA. MGPA is stable to boiling, to guanidine hydrochloride treatment,to O-glycanase treatment; to digestion with trypsin; and to multiplecycles of lyophilization or freeze thawing.

EXAMPLE 5 Amino Acid Sequence and Cloning of MGPA

Pure MGPA is sequenced using conventional microsequencing techniquessuitable for studies of picomolar amounts of protein. Amino terminalsequence is complemented with sequences of peptides derived by peptidaseor cyanogen bromide digestion, separated by reverse phase HPLC or SDSgel electrophoresis and electroelution. The amino acid sequence of MGPAis screened for uniqueness using the PIR data banks.

The degree of glycosylation is assessed by amino acid/amino sugardetermination and by molecular weight shift on SDS gel of iodinated MGPAafter digestion by a panel of glycosidases including neuraminidase,sialidase, F-endoglycosidase, and G-endoglycosidase.

The generation of large amounts of MGPA for extensive physical,chemical, cell biological and preclinical studies is optimally performedby molecular cloning of the MGPA gene and expression of this gene orcorresponding cDNA in any one of a variety of host/vector systems.

To obtain the cDNA for MGPA, probes consisting of pools ofoligonucleotides or unique oligonucleotides are designed from thetryptic sequences according to the method of R. Lathe, J. Mol. Biol.,183(1):1-12 (1985). The oligonucleotide probes are synthesized on anautomated DNA synthesizer.

Because the genetic code is degenerate (more than one codon can code forthe same amino acid) a mixture of oligonucleotides are synthesized thatcontain all possible nucleotide sequences encoding the amino acidsequence of the selected tryptic fragment or portion thereof. It may bepossible in some cases to reduce the number of oligonucleotides in theprobe mixture based on codon usage because some codons are rarely usedin eukaryotic genes, and because of the relative infrequency of thedinucleotide CpG in eukaryotic coding sequences [see J. J. Toole et al,Nature, 312:342-347 (1984)]. The regions of the amino acid sequencesused for probe design are chosen by avoiding highly degenerate codonswhere possible. The oligonucleotides are synthesized on an automated DNAsynthesizer and the probes are then radioactively labelled withpolynucleotide kinase and ³² p-ATP.

cDNA is then synthesized from polyadenylated RNA from a human cell line,e.g., one of the above mentioned cell sources of MGPA using eitherconventional cloning technology or polymerase chain reaction technology.The cDNA library may be cloned into lambda ZAP [Stratagene CloningSystems, La Jolla, CA] or other suitable vectors using establishedtechniques Calif. (see Toole et al cited above). Recombinants from thislibrary are plated and duplicate nitrocellulose replicas made of theplates. The oligonucleotides are kinased with ³² P gamma ATP andhybridized to the replicas at a temperature predicted from the lengthand base composition of the probes [See, J. Singer-Sam et al, Proc.Nat'l. Acad. Sci. USA. 80:802-806 (1983) and S. V. Suggs et al, in"Developmental Biology Using Purified Genes", ICN-UCLA Symposium onMolecular and Cellular Biology, eds. Brown D. D. and Fox, C. F.(Academic, N.Y.), Vol. 23, pp. 683-693 (1981)] in standard hybridizationsolution overnight. The filters are then washed in 0.5XSSC at the sametemperature until the background radioactivity is lowered to anacceptable level to allow autoradiography. Alternatively, thehybridization and washes may be performed in the presence oftetraalkylammonium salt solution [See K. A. Jacobs et al, Nucl. AcidsRes., 16:4637-4650 (1988)]. Duplicate positives are plaque purified.

A complete or partial cDNA sequence is obtained by this method. Thissequence may be used optionally as a probe to rescreen the library toobtain full length cDNAs. It is also possible that partial cDNAs mayyield an active MGPA fragment.

Alternatively, the MGPA gene may be isolated from a human genomiclibrary (available from Stratagene) in λ Zap using the oligonucleotidehybridization probes described above. The genomic MGPA clone isexpressed directly in mammalian cells or used to isolate a cDNA. In thelatter case, the MGPA gene is used as a hybridization probe to identifya source of MGPA in RNA. Alternatively, the MGPA gene is expressedtransiently in COS1 cells to generate an MGPA mRNA that can be used togenerate a cDNA.

EXAMPLE 6 Expression of Recombinant Human MGPA

To produce MGPA or an active fragment thereof, the cDNA encoding it istransferred into an appropriate expression vector, of which numeroustypes are known in the art for human, insect, yeast, fungal andbacterial expression, by standard molecular biology techniques.

One such vector for mammalian cells is pXM [Y. C. Yang et al, Cell,47:3-10 (1986)]. This vector contains the SV40 origin of replication andenhancer, the adenovirus major late promoter, a cDNA copy of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,in appropriate relationships to direct the high level expression of thedesired cDNA in mammalian cells [See, e.g., Kaufman, Proc. Natl. Acad.Sci. USA, 82:689-693 (1985)]. The pXM vector is linearized with theendonuclease enzyme XhoI and subsequently ligated in equimolar amountseparately to the cDNA encoding MGPA that has been previously modifiedby addition of synthetic oligonucleotides that generate Xho Icomplementary ends to generate constructs for expression.

Another vector for mammalian expression is pEMC2Bl. This vector may bederived from pMT2pc which has been deposited with the American TypeCulture Collection (ATCC), Rockville, Md. (USA) under Accession NumberATCC 40348. The DNA is linearized by digestion of the plasmid with PstI.The DNA is then blunted using T₄ DNA polymerase. An oligonucleotide 5'TGCAGGCGAGCCTGAATTCCTCGA 3' is then ligated into the DNA, recreating thePstI site at the 5' end and adding an EcoRI site and XhoI site beforethe ATG of the DHFR cDNA. This plasmid is called pMT21. pMT21 is cutwith EcoRI and XhoI which cleaves the plasmid at two adjacent cloningsites. An EMCV fragment of 508 base pairs is cut from pMT₂ ECAT₁ [S. K.Jong et al, J. Virol., 63:1651-1660 (1989)] with the restriction enzymesEcoRI and TaqαI. A pair of oligonucleotides 68 nucleotides in length aresynthesized to duplicate the EMCV sequence up to the ATG. The ATG ischanged to an ATT, and a C is added, creating a XhoI site at the 3' end.A TaqαI site is situated at the 5' end. The sequences of theoligonucleotides are: 5'CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT GAAAACACGATTGC 3'and its complementary strand.

Ligation of the pMT21 EcoRI-to-XhoI fragment to the EMCV EcoRI-to-TaqoIfragment and to the TaqoI/XhoI oligonucleotides produces the vectorpEMC2B1. This vector contains the SV40 origin of replication andenhancer, the adenovirus major late promoter, a cDNA copy of themajority of the adenovirus tripartite leader sequence, a small hybridintervening sequence, an SV40 polyadenylation signal and the adenovirusVA I gene, DHFR and β-lactamase markers and an EMC sequence, inappropriate relationships to direct the high level expression of thedesired cDNA in mammalian cells. The EMC2B1 vector is linearized withthe endonuclease enzyme EcoRI and subsequently ligated in equimolaramount separately to the cDNA encoding MGPA that has been previouslymodified by addition of synthetic oligonucleotides that generate EcoRIcomplementary ends to generate constructs for expression. Theseconstructs can be expressed in various hosts with appropriate vectors.

The vector is then introduced into appropriate host cells byconventional genetic engineering techniques. The transformed cells arecultured and the expressed MGPA is recovered and purified from theculture medium using standard techniques.

a. Mammalian Cell Expression

To obtain expression of the MGPA protein, the pXM construct containingthe cDNA is mixed and transfected into COS cells, for example. Theconditioned medium from the transfected COS cells contains MGPAbiological activity as measured in the megakaryocyte growth promotingassay of Example 1.

The mammalian cell expression vectors described herein may besynthesized by techniques well known to those skilled in this art. Oneskilled in the art can also construct other mammalian expression vectorscomparable to the pXM vector by, e.g., inserting the DNA sequence of theMGPA from the plasmid with appropriate enzymes and employing well-knownrecombinant genetic engineering techniques and other known vectors, suchas pJL3 and pJL4 [Gough et al., EMBO J., 4:645-653 (1985)] and pMT2(starting with pMT2-VWF, ATCC #67122; see PCT applicationPCT/US87/00033).

Mammalian host cells other than COS cells may also be employed in MGPAexpression. For example, preferably for stable integration of the vectorDNA, and for subsequent amplification of the integrated vector DNA, bothby conventional methods, CHO cells may be employed as a mammalian hostcell of choice.

Once the vectors and host cells are selected and transformed, stabletransformants are then screened for expression of the product bystandard immunological, biological or enzymatic assays, such as thosedescribed above in Examples 1 and 4. The presence of the DNA and mRNAencoding the MGPA polypeptides may be detected by standard proceduressuch as Southern and Northern blotting. Transient expression of the DNAencoding the polypeptides during the several days after introduction ofthe expression vector DNA into suitable host cells is measured withoutselection by bioactivity or immunologic assay of the proteins in theculture medium.

b. Bacterial Exoression Systems

Similarly, one skilled in the art could manipulate the sequencesencoding the MGPA polypeptide by eliminating any human regulatorysequences flanking the coding sequences and inserting bacterialregulatory sequences to create bacterial vectors for intracellular orextracellular expression of the MGPA polypeptide of the invention bybacterial cells. The DNA encoding the polypeptides may be furthermodified to contain different codons to optimize bacterial expression asis known in the art. Preferably the sequences encoding the mature MGPAare operatively linked in-frame to nucleotide sequences encoding asecretory leader polypeptide permitting bacterial expression, secretionand processing of the mature MGPA polypeptides, also by methods known inthe art. The expression of MGPA in E. coli using such secretion systemsis expected to result in the secretion of the active polypeptide. Thisapproach has yielded active chimeric antibody fragments [See, e.g.,Bitter et al, Science, 240:1041-1043 (1983)]. Alternatively, the MGPAmay be expressed as a cytoplasmic protein in E. coli. In this case, themolecule would most likely have to be refolded after completedenaturation with guanidine hydrochloride, a process also known in theart. For procedures for isolation and refolding of intracellularlyexpressed proteins, see, for example, U.S. Pat. No. 4,512,922.

The compounds expressed through either route in bacterial host cells maythen be recovered, purified, and/or characterized with respect tophysicochemical, biochemical and/or clinical parameters, all by knownmethods.

c. Insect or Yeast Cell Expression

Similar manipulations can be performed for the construction of an insectvector for expression of MGPA polypeptides in insect cells [See, e.g.,procedures described in published European patent application 155,476].

Similarly yeast vectors are constructed employing yeast regulatorysequences to express cDNA encoding the precursor, in yeast cells toyield secreted extracellular active MGPA. Alternatively the polypeptidemay be expressed intracellularly in yeast, the polypeptide isolated andrefolded to yield active MGPA. [See, e.g., procedures described inpublished PCT application WO 86/00639 and European patent application EP123,289. ]

EXAMPLE 7 Construction of CHO Cell Lines Expressing High Levels of MGPA

One method for producing high levels of the MGPA polypeptide of theinvention from mammalian cells involves the construction of cellscontaining multiple copies of the cDNA encoding the MGPA.

The cDNA is co-transfected with an amplifiable marker, e.g., the DHFRgene for which cells containing increasing concentrations ofmethotrexate (MTX) according to the procedures of Kaufman and Sharp, J.Mol. Biol., (1982) supra. This approach can be employed with a number ofdifferent cell types.

For example, the pXM vector or the pEMC2B1 vector containing the MGPAgene in operative association with other plasmid sequences enablingexpression thereof is introduced into DHER-deficient CHO cells,DUKX-BII, along with a DHFR expression plasmid such as pAdD26SVpA3[Kaufman, Proc. Natl. Acad. Sci. USA. 82:689-693 (1985)] by eithercalcium phosphate coprecipitation or protoplast fusion, followed bytransfection. The MGPA gene and marker gene may be on a single plasmidor on two plasmids for transfection. DHFR expressing transformants areselected for growth in alpha media with dialyzed fetal calf serum.Transformants are checked for expression of MGPA by bioassay,immunoassay or RNA blotting and positive pools are subsequently selectedfor amplification by growth in increasing concentrations of MTX(sequential steps in 0.02, 0.2, 1.0 and 5 uM MTX) as described inKaufman et al., Mol. Cell Biol., 5:1750 (1983). The amplified lines arecloned, and MGPA expression is monitored by the MGPA assay of Example I.MGPA expression is expected to increase with increasing levels of MTXresistance.

In any of the expression systems described above, the resulting celllines can be further amplified by appropriate drug selection, resultingcell lines recloned and the level of expression assessed using the MGPAassay described in Example I.

The MGPA expressing CHO cell lines can be adapted to growth inserum-free medium. Homogeneous MGPA can be isolated from conditionedmedium from the cell line using methods familiar in the art, includingtechniques such as lectin-affinity chromatography, reverse phase HPLC,FPLC and the like.

EXAMPLE 8 Antibodies to MGPA

Purified MGPA and synthesized peptides from its sequence will be used toproduce polyclonal antisera in rabbits and monoclonal antibodies in miceusing conventional methods to generate ahtibodies to detect andquantitate MGPA in clinical samples, and to screen for antibodies thatblock MGPA for therapeutic use.

Mice and rabbits are immunized with pure urinary MGPA or with individualpeptide sequences 12-20 amino acids in length. Serum from the animals iscollected weekly and tested for reactivity by Western blotting to wholeMGPA. Wells with immunoreactivity on Western blot are expanded andsubcloned. Individual antibodies are tested for their affinity to MGPA,and for their ability to block the action of MGPA by the direct additionof supernatant to marrow cell cultures where both polyclonal rabbitantisera [anti-IL-3, anti-GM-CSF] and monoclonal murine antisera[anti-erythropoietin] block the effect of relevant growth factors.

These monoclonal and polyclonal antibodies are used in radioimmunoassaysfor evaluation of patient samples. Measurement of 0.5-500 units/mlshould be easily performed in plasma or urine (timed out-put) withmonoclonals of average affinity.

A series of antibodies to MGPA is useful in detecting and blocking MGPA,and determining its receptor binding site. Both polyclonal antisera anda series of monoclonals against MGPA peptides are useful in establishingthe immunologic identity of plasma and urinary MGPA.

The foregoing description details presently preferred embodiments of theinvention. Numerous modifications and variations in practice of thisinvention are expected to occur to those skilled in the art. Suchmodifications and variations are encompassed within the followingclaims.

What is claimed is:
 1. Isolated megakaryocyte growth promoting activity(MGPA) protein having the following characteristics:(1) an apparentmolecular weight of approximately 40-50 l kD as determined by SDS-PAGE;(2) a specific activity in a radioimmunological megakaryocyte growthpromoting assay of approximately 1×10⁶ to 1.1×10⁷ units per mgpolypeptide; and (3) an ability to support megakaryocyte colony growthin a human bone marrow liquid culture assay in the presence of IL-3. 2.Isolated megakaryocyte growth promoting (MGPA) activity protein havingthe following characteristics:(1) an apparent molecular weight ofapproximately 40-50 kD as determined by SDA-PAGE; (2) a specificactivity in a radioimmunological megakaryocyte growth promoting assay ofgreater than 1×10⁶ units per mg polypeptide; and (3) an ability tosupport megakaryocyte growth in a human bone marrow liquid culture assayin the presence of IL-3.
 3. The protein according to claim 2 having oneor more of the following characteristics:(1) an apparent molecularweight of approximately 45 kd as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis; (2) an apparent molecular weight ofapproximately 40-50 kd as determined by gel filtration chromatography;(3) the inability to bind to Wheat Germlectin; (4) the ability to bindto a Pharmacia Mono S cation exchange column at acidic pH; (5) theinability to bind to an anion exchange resin at neutral pH; (6) presencein 30-50% ammonium sulphate precipitate of thrombocytopenic patientplasma.
 4. The protein according to claim 2 produced by culturing a cellline transformed with a DNA sequence encoding expression of MGPA inoperative association with an expression control sequence therefor.
 5. Acomposition comprising the MGPA of claim 2 in a pharmaceuticallyeffective vehicle.
 6. The composition according to claim 5 furthercomprising a cytokine, hematopoietin, growth factor, meg-CSF orthrombopoietin-like factor.
 7. The composition according to claim 6where said cytokine is selected from the group consisting of G-CSF,CSF-1, GM-CSF, IL-1, IL-3, IL-4, meg-CSF, erythropoietin, IL-6, TPO,M-CSF and IL-7.
 8. The composition according to claim 7 wherein saidcytokine is IL-3 and GM-CSF.